This invention relates to a method for the manufacture of maltose by use of enzymes which are produced by a single microorganism. More particularly, the present invention concerns a method for direct manufacture of maltose from starch by use of a microorganism capable of simultaneously producing .beta.-amylase and .alpha.-1,6-glucosidase.
Heretofore, .beta.-amylase (.alpha.-1,4-glucan maltohydrolase) has been found to occur mostly in malt and soybean. Commercially, these are used as sources for the enzyme today. Besides, microorganisms of genus Bacillus have been known to be capable of producing a similar enzyme. For example, Kneen et al. discovered in 1946 that Bacillus polymyxa produces .beta. -amylase (Archives of Biochemistry, Vol. 10, p 41 (1946)) and, in 1948, Rose made a more detailed report on the enzymatic property of .beta.-amylase produced by the strain of said microorganism (Archives of Biochemistry, Vol. 16, p 349 (1948)). After that, Higashihara et al. reported that Bacillus megaterium produces .beta. -amylase (Japan Agricultural Chemical Society, Abstracts of Lectures at the 1971 Annual Meeting, p 212 and Amylase Symposium, Vol. 6, p 39 (1971)). It was reported that this enzyme is identical with the .beta.-amylase produced by Bacillus polymyxa (Japan Agricultural Chemical Society, Abstracts of Lectures at the 1972 Annual Meeting p 86.
On the other hand, as concerns .alpha.-1,6-glucosidase of starch, many reports so far made treat this enzyme as isoamylase or pullulanase. To be specific, isoamylase was first discovered to occur in yeasts by Maruo, Kobayashi et al. (Bunji Maruo and Tsuneo Kobayashi, Journal of Japan Agricultural Chemical Society, Vol. 23, pp 115 and 120 (1949). This enzyme has since been found to occur in higher plants (the enzyme from such source is referred to as R-enzyme) and in microorganisms of genus Pseudomonas (Japanese Patent Publication No. 16788/1970). More recently, a report was made to the effect that thermophilic Bacillus stearothermophilas produces a thermophilic isoamylase whose optimum working temperature is 65.degree. to 67.5.degree. C (Japan Agricultural Chemical Society, Abstracts of Lectures at the 1972 Annual Meeting, p 88 and Japanese Patent Disclosure No. 91272/1973).
Pullulanase was found by Bender in 1959 to occur in Aerobacter aerogenes as an enzyme capable of hydrolyzing the polysaccharide pullulan produced by Pullularia pullulan. It hydrolyzes the .alpha.-1,6-glucosidasic linkage of pullulan to give rise to maltotriose (Biochem. Biophys. Acta, Vol. 36, p. 309 (1959) and Japanese Patent Publication No. 7559/1971). This enzyme also hydrolyzes the .alpha.-1,6-glycosidasic linkage such as of amylopectin and glycogen. It has since been reported that enzymes of such description are also produced by microorganisms such as Escherichia intermedia (Ueda et al. Applied Microbiology, Vol. 15, p. 492 (1967)) and Streptomyces mites (Ueda et al., Journal of Fermentation Technology, Vol. 49, p 552 (1971)).
As described above, independent production of .beta.-amylase and .alpha.-1,6-glucosidase has been reported in numerous articles. However, no report has ever been made concerning a microorganism which produces both .beta.-amylase and .alpha.-1,6-glucosidase at the same time.
Therefore, it had been necessary to manufacture both enzymes by cultivating two different microorganisms separately. Besides, on account of the difference in the reaction pH and reaction temperature of the two enzymes, it has been difficult to react the enzymes upon starch at the same time. Therefore, the manufacture of maltose has heretofore been carried out by a two-step process which generally comprises first causing .alpha.-1,6-glucosidase to react upon starch to produce a straight-chain amylose-like substance and subsequently having .beta.-amylase react thereon to effect conversion to maltose.